Millipore Centriprep 50k User Manual

User Guide
Centriprep
®
Centrifugal Filter Devices
Centriprep® 3K and 50K devices for research
use only; not for use in diagnostic procedures
Introduction
Centriprep® centrifugal filter devices are disposable ultrafiltration devices used for purifying,
concentrating, desalting, and filtering biological samples in the 2–15 mL volume range. These ready-
to-use ultrafiltration devices are designed for operation in most centrifuges that can accommodate
50 mL centrifuge tubes. They are easy to use and offer a high flow rate. The Centriprep® device consists
of a sample container with a twist-lock cap, a filtrate collector containing a low adsorptive Ultracel®
regenerated cellulose membrane, and an air-seal cap for sample isolation.
The Centriprep® device design allows filtration and fine particle sedimentation to occur at the same time
without clogging the membrane. With a Centriprep® 50K device, a 15 mL sample can be concentrated to
0.6 mL in just 20 minutes and concentration of smaller volumes takes even less time. High concentration
factors are achieved because of the membrane's low adsorptivity, and greater than 90% recovery of retained
macromolecular solutes is typical. A built-in deadstop provides a final concentrated sample volume of
0.5–0.6 mL.
The Centriprep® product line includes 4 different molecular weight cutoffs (MWCO):
● Centriprep® 3K device — 3,000 MWCO (yellow membrane support)
● Centriprep® 10K device — 10,000 MWCO (green membrane support)
● Centriprep® 30K device — 30,000 MWCO (colorless membrane support)
● Centriprep® 50K device — 50,000 MWCO (pink membrane support)
NOTE: Centriprep® 10K and 30K devices are the only Centriprep® devices intended for in vitro diagnostic
use. For information on these devices, go to www.millipore.com/centrifugal_ivd_userguide.
Applications
Centriprep® 3K centrifugal filter devices:
● Concentrating and desalting oligonucleotides, peptides, growth factors, and small proteins.
Centriprep® 10K, 30K, and 50K centrifugal filter devices:
● Concentrating and desalting column eluates and gradient fractions containing proteins, enzymes,
and antibodies
● Recovering biomolecules from cell culture supernatants, lysates, extracts, or other biological samples
● Purifying low-molecular-weight components (e.g., amino acids and antibiotics) from physiological
fluids, cell culture media, or fermentation broths.
Centriprep® Device Components
Air Seal Cap
Twist-Lock Cap
Vent Groove
Filtrate Collector Shoulder
Filtrate Collector
Membrane Support
Sample Container
Fill Line
Required Equipment
Centriprep® devices can be spun in a variable speed centrifuge with either a swinging-bucket or fixed-
angle rotor and carrier that can properly accommodate 50 mL centrifuge tubes. The centrifuge must be
capable of 500–3,000 × g. For a listing of compatible centrifuges and rotors, see www.millipore.com/
rotorguide.
Limitations
With fixed-angle rotors, polarization control may be adversely affected at low g-forces (below 2,000 × g).
Depending on sample composition and solute concentration, filtrate flow may be reduced. When using
either type of centrifuge, do not exceed the following limits because excessive g-force may result in
leakage or damage to the device.
Device Maximum Centrifugal Force
Centriprep® 3K
Centriprep® 50K
Device Storage and Shelf Life
For best results, store Centriprep® devices at 15–30 °C. Performance may be adversely affected if the
devices are stored at extreme temperatures (i.e., below 0 °C or above 40 °C). Shelf life is three years from
date of manufacture.
Rinsing Before Use
The membranes in Centriprep® centrifugal filter devices contain trace amounts of glycerine. If this inter-
feres with analysis, fill the device with approximately 15 mL of buffer or deionized water and centrifuge
until the first equilibration point is reached. Decant filtrate and concentrate. Repeat as necessary until no
more interference is noted. Once wet, membranes must be kept wet to remain functional.
Membrane and Device Appearance
● Occasionally, an unused Centriprep® device may have a few drops of moisture in the filtrate collector
or sample container. This is due to condensation of moisture in the membrane and does not affect
performance.
● After centrifugation, a small amount of filtrate (approximately 0.5 mL) may remain in the area
between the membrane and the membrane support of the Centriprep® device. This is normal and does
not affect device operation or performance.
● The Ultracel® membrane used in the Centriprep® centrifugal filter devices may appear slightly wrinkled
after spinning.
Centrifugal Filter Device Reuse
Centriprep® devices are intended for single use only. Attempts to clean the device may damage the
membrane surface or the seal, possibly compromising the unit's integrity and thereby affecting
performance.
Principle of Operation
To begin operation, the sample container's twist-lock cap is loosened and the filtrate collector is removed.
Sample is added, then the filtrate collector is carefully reinserted back into the sample container (displacing
solution) and the cap locked, sealing the device. The assembled centrifugal filter device is centrifuged at
1,000–3,000 × g, depending on the membrane MWCO used.
Immersing the filtrate collector in the sample solution creates a slight hydrostatic pressure differential
that exerts an upward buoyancy force on the membrane at the filtrate collector bottom. By itself, the
hydrostatic pressure exerted by the displaced solution is too weak to produce ultrafiltration. Centrifugation
increases this pressure, forcing low molecular weight materials and solvent through the membrane into
the filtrate collector. Solutes with molecular weights above the membrane MWCO remain in the sample
container and become increasingly concentrated as the operation continues. Ultrafiltration occurs in the
direction opposite the centrifugal force vector.
Centrifugal Force
Filtration
Polarization
Swinging-Bucket Rotor
The pressure differential created by the raised solution level is important in centrifugal filter device
operation. During centrifugation, the sample solution meniscus falls as the filtrate meniscus rises. In the
process, the filtrate collector loses its buoyancy and sinks to the bottom of the sample container, permitting
maximum filtrate collection. Eventually, an equilibrium is reached where the menisci are at equal heights.
Filtration then stops, since the hydrostatic pressure difference is now zero.
If further concentration is required, decant the filtrate before spinning the device for a second time.
Decanting re-establishes the pressure differential between the sample meniscus and the filtrate meniscus,
allowing filtration to resume. Filtration continues until a new equilibrium point is achieved. Note that the
pressure differential is constantly changing during operation as the menisci rise and fall.
Centriprep® centrifugal filter devices are designed to maintain high flow rates by minimizing solute
build-up on the membrane during operation. Centrifugal force causes dense materials to sink away from
the membrane to the sample container bottom. Because the Centriprep® membrane "floats" above this
polarization layer, the full surface of the membrane is used at all times, and filtrate flow is unrestricted.
This is particularly beneficial when working with suspensions, which can easily clog or foul a membrane.
3,000 × g
1,500 × g
Layer
Fixed-Angle Rotor